DMD genotype correlations from the Duchenne Registry: Endogenous exon skipping is a factor in prolonged ambulation for individuals with a defined mutation subtype.

Antisense oligonucleotide (AON)-mediated exon skipping is an emerging therapeutic for individuals with Duchenne muscular dystrophy (DMD). Skipping of exons adjacent to common exon deletions in DMD using AONs can produce in-frame transcripts and functional protein. Targeted skipping of DMD exons 8, 44, 45, 50, 51, 52, 53, and 55 is predicted to benefit 47% of affected individuals. We observed a correlation between mutation subgroups and age at loss of ambulation in the Duchenne Registry, a large database of phenotypic and genetic data for DMD (N = 765). Males amenable to exon 44 (N = 74) and exon 8 skipping (N = 18) showed prolonged ambulation compared to other exon skip groups and nonsense mutations (P = 0.035 and P < 0.01, respectively). In particular, exon 45 deletions were associated with prolonged age at loss of ambulation relative to the rest of the exon 44 skip amenable cohort and other DMD mutations. Exon 3-7 deletions also showed prolonged ambulation relative to all other exon 8 skippable mutations. Cultured myotubes from DMD patients with deletions of exons 3-7 or exon 45 showed higher endogenous skipping than other mutations, providing a potential biological rationale for our observations. These results highlight the utility of aggregating phenotypic and genotypic data for rare pediatric diseases to reveal progression differences, identify potentially confounding factors, and probe molecular mechanisms that may affect disease severity.

Family-specific aggregation of lipid GWAS variants confers the susceptibility to familial hypercholesterolemia in a large Austrian family.

BACKGROUND AND AIMS: Hypercholesterolemia confers susceptibility to cardiovascular disease (CVD). Both serum total cholesterol (TC) and LDL-cholesterol (LDL-C) exhibit a strong genetic component (heritability estimates 0.41-0.50). However, a large part of this heritability cannot be explained by the variants identified in recent extensive genome-wide association studies (GWAS) on lipids. Our aim was to find genetic causes leading to high LDL-C levels and ultimately CVD in a large Austrian family presenting with what appears to be autosomal dominant inheritance for familial hypercholesterolemia (FH). METHODS: We utilized linkage analysis followed by whole-exome sequencing and genetic risk score analysis using an Austrian multi-generational family with various dyslipidemias, including elevated TC and LDL-C, and one family branch with elevated lipoprotein (a) (Lp(a)). RESULTS: We did not find evidence for genome-wide significant linkage for LDL-C or apparent causative variants in the known FH genes rather, we discovered a particular family-specific combination of nine GWAS LDL-C SNPs (p = 0.02 by permutation), and putative less severe familial hypercholesterolemia mutations in the LDLR and APOB genes in a subset of the affected family members. Separately, high Lp(a) levels observed in one branch of the family were explained primarily by the LPA locus, including short (<23) Kringle IV repeats and rs3798220. CONCLUSIONS: Taken together, some forms of FH may be explained by family-specific combinations of LDL-C GWAS SNPs.

Rare Inherited and De Novo CNVs Reveal Complex Contributions to ASD Risk in Multiplex Families.

Rare mutations, including copy-number variants (CNVs), contribute significantly to autism spectrum disorder (ASD) risk. Although their importance has been established in families with only one affected child (simplex families), the contribution of both de novo and inherited CNVs to ASD in families with multiple affected individuals (multiplex families) is less well understood. We analyzed 1,532 families from the Autism Genetic Resource Exchange (AGRE) to assess the impact of de novo and rare CNVs on ASD risk in multiplex families. We observed a higher burden of large, rare CNVs, including inherited events, in individuals with ASD than in their unaffected siblings (odds ratio [OR] = 1.7), but the rate of de novo events was significantly lower than in simplex families. In previously characterized ASD risk loci, we identified 49 CNVs, comprising 24 inherited events, 19 de novo events, and 6 events of unknown inheritance, a significant enrichment in affected versus control individuals (OR = 3.3). In 21 of the 30 families (71%) in whom at least one affected sibling harbored an established ASD major risk CNV, including five families harboring inherited CNVs, the CNV was not shared by all affected siblings, indicating that other risk factors are contributing. We also identified a rare risk locus for ASD and language delay at chromosomal region 2q24 (implicating NR4A2) and another lower-penetrance locus involving inherited deletions and duplications of WWOX. The genetic architecture in multiplex families differs from that in simplex families and is complex, warranting more complete genetic characterization of larger multiplex ASD cohorts.

Genome-wide linkage and association analyses implicate FASN in predisposition to Uterine Leiomyomata.

Uterine leiomyomata (UL), the most prevalent pelvic tumors in women of reproductive age, pose a major public health problem given their high frequency, associated morbidities, and most common indication for hysterectomies. A genetic component to UL predisposition is supported by analyses of ethnic predisposition, twin studies, and familial aggregation. A genome-wide SNP linkage panel was genotyped and analyzed in 261 white UL-affected sister-pair families from the Finding Genes for Fibroids study. Two significant linkage regions were detected in 10p11 (LOD = 4.15) and 3p21 (LOD = 3.73), and five additional linkage regions were identified with LOD scores > 2.00 in 2q37, 5p13, 11p15, 12q14, and 17q25. Genome-wide association studies were performed in two independent cohorts of white women, and a meta-analysis was conducted. One SNP (rs4247357) was identified with a p value (p = 3.05 × 10(-8)) that reached genome-wide significance (odds ratio = 1.299). The candidate SNP is under a linkage peak and in a block of linkage disequilibrium in 17q25.3, which spans fatty acid synthase (FASN), coiled-coil-domain-containing 57 (CCDC57), and solute-carrier family 16, member 3 (SLC16A3). By tissue microarray immunohistochemistry, we found elevated (3-fold) FAS levels in UL-affected tissue compared to matched myometrial tissue. FAS transcripts and/or protein levels are upregulated in various neoplasms and implicated in tumor cell survival. FASN represents the initial UL risk allele identified in white women by a genome-wide, unbiased approach and opens a path to management and potential therapeutic intervention.

Identification of two common variants contributing to serum apolipoprotein B levels in Mexicans.

BACKGROUND AND PURPOSE: Although the Mexican population has a high predisposition to dyslipidemias and premature coronary artery disease, this population is underinvestigated for the genetic factors conferring the high susceptibility. This study attempted to determine these genetic factors. METHODS AND RESULTS: First, we investigated apolipoprotein B (apoB) levels in Mexican extended families with familial combined hyperlipidemia using a two-step testing strategy. In the screening step, we screened 5721 single-nucleotide polymorphisms (SNPs) for linkage signals with apoB. In the test step, we analyzed the 130 SNPs residing in regions of suggestive linkage signals for association with apoB. We identified significant associations with two SNPs (ie, rs1424032 [P=6.07x10(-6)] and rs1349411 [P=2.72x10(-4)]) that surpassed the significance level for the number of tests performed in the test step (P<3.84x10(-4)). Second, these SNPs were tested for replication in Mexican hyperlipidemic case-control samples. The same risk alleles as in the families with familial combined hyperlipidemia were significantly associated (P<0.05) with apoB in the case-control samples. The rs1349411 resides near the apoB messenger RNA editing enzyme (APOBEC1) involved in the processing of APOB messenger RNA in the small intestine. The rs1424032 resides in a highly conserved noncoding region predicted to function as a regulatory element. CONCLUSIONS: We identified two novel variants, rs1349411 and rs1424032, for serum apoB levels in Mexicans.

WW-domain-containing oxidoreductase is associated with low plasma HDL-C levels.

Low serum HDL-cholesterol (HDL-C) is a major risk factor for coronary artery disease. We performed targeted genotyping of a 12.4 Mb linked region on 16q to test for association with low HDL-C by using a regional-tag SNP strategy. We identified one SNP, rs2548861, in the WW-domain-containing oxidoreductase (WWOX) gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent (p = 6.9 x 10(-7)). We extended our investigation to the population level by using two independent unascertained population-based Finnish cohorts, the cross-sectional METSIM cohort of 4,463 males and the prospective Young Finns cohort of 2,265 subjects. The combined analysis provided p = 4 x 10(-4) to 2 x 10(-5). Importantly, in the prospective cohort, we observed a significant longitudinal association of rs2548861 with HDL-C levels obtained at four different time points over 21 years (p = 0.003), and the T risk allele explained 1.5% of the variance in HDL-C levels. The rs2548861 resides in a highly conserved region in intron 8 of WWOX. Results from our in vitro reporter assay and electrophoretic mobility-shift assay demonstrate that this region functions as a cis-regulatory element whose associated rs2548861 SNP has a specific allelic effect and that the region forms an allele-specific DNA-nuclear-factor complex. In conclusion, analyses of 9,798 subjects show significant association between HDL-C and a WWOX variant with an allele-specific cis-regulatory function.

Interactive effects of cadmium and all-trans-retinoic acid on the induction of forelimb ectrodactyly in C57BL/6 mice.

BACKGROUND: Most toxicological studies have tested single chemical agents at relatively high doses, and fewer studies have addressed the toxic effects of chemical interactions. It is important to understand the toxicity of chemical mixtures in order to assess the more realistic risks of environmental and occupational exposures. A number of chemicals are known to induce a predominantly postaxial forelimb ectrodactyly in C57BL/6 mice, including acetazolamide, ethanol, cadmium, valproic acid, carbon dioxide, dimethadione, phenytoin, and 13-cis-retinoic acid and all-trans-retinoic acid (RA). In the present study, the interactive effects of coadministration of cadmium and RA on developing limbs were investigated. METHODS: Pregnant C57BL/6 mice were treated with different intraperitoneal (IP) doses of cadmium chloride (CdCl2) and/or RA on gestational day (GD) 9.5, and fetuses were collected on GD 18 and double stained for examination of skeletal defects. RESULTS: When RA was given simultaneously with cadmium, a significant increase in the incidence and severity of forelimb ectrodactyly (predominantly postaxial) was observed compared to the results with corresponding doses of cadmium or RA alone. When mice were exposed to subthreshold doses of both cadmium (0.5 mg/kg) and RA (1 mg/kg), the combined treatment exceeded the threshold, resulting in forelimb ectrodactyly in 19% of the fetuses. Moreover, coadministration of cadmium and RA at doses exceeding the respective thresholds showed a synergistic effect, that is, 92% of fetuses were found with the forelimb defect as opposed to 10% if the response were additive. CONCLUSIONS: The findings demonstrate that concurrent exposure to these teratogens can have a synergistic effect and that subteratogenic doses may combine to exceed a threshold.

Cross-species analyses implicate Lipin 1 involvement in human glucose metabolism.

Recent studies in the mouse have demonstrated that variations in lipin expression levels in adipose tissue have marked effects on adipose tissue mass and insulin sensitivity. In the mouse, lipin deficiency prevents normal adipose tissue development, resulting in lipodystrophy and insulin resistance, whereas excess lipin levels promote fat accumulation and insulin sensitivity. Here, we investigated the effects of genetic variation in lipin levels on glucose homeostasis across species by analyzing lipin transcript levels in human and mouse adipose tissues. A strong negative correlation was observed between lipin mRNA levels and fasting glucose and insulin levels, as well as an indicator of insulin resistance (HOMA-IR), in both mice and humans. We subsequently analyzed the allelic diversity of the LPIN1 gene in dyslipidemic Finnish families, as well as in a case-control sample of obese (n = 477) and lean (n = 821) individuals. Alleles were defined by genotyping seven single nucleotide polymorphisms (SNPs) of the critical DNA region over the LPIN1 gene. Intragenic SNPs and corresponding allelic haplotypes exhibited associations with serum insulin levels and body mass index (P = 0.002-0.04). Both the expression levels in adipose tissue across species and genetic data in human study samples highlight the importance of lipin in glucose homeostasis and imply that allelic variants of this gene have significance in human metabolic traits.

Search for autism loci by combined analysis of Autism Genetic Resource Exchange and Finnish families.

OBJECTIVE: Several genome-wide screens have been performed in autism spectrum disorders resulting in the identification of numerous putative susceptibility loci. Analyses of pooled primary data should result in an increased sample size and the different study samples have a potential to strengthen the evidence for some earlier identified loci, reveal novel loci, and even to provide information of the general significance of the locus. The objective of this study was to search for potential susceptibility loci for autism, which are supported by two independent samples. METHODS: We performed a combined analysis of the primary genome scan data of the Autism Genetic Resource Exchange (AGRE) and Finnish autism samples to reveal susceptibility loci potentially shared by these study samples. RESULTS: In the initial combined data analysis, the best loci (p < 0.05) were observed at 1p12-q25, 3p24-26, 4q21-31, 5p15-q12, 6q14-21, 7q33-36, 8q22-24, 17p12-q21, and 19p13-q13. The combined analysis of Finnish and AGRE families showed the most promising shared locus on 3p24-26 with nonparametric logarithm of odds (NPL) score of 2.20 (p = 0.011). The combined data analysis did not provide increased linkage evidence for the earlier identified loci on 3q25-27 or 17p12-q21. However, the 17p12-q21 locus remained promising also in the combined sample (NPL(all) =2.38, p = 0.0076). INTERPRETATION: Our study of 314 autism families highlights the importance of further analyses on 3p24-26 locus involving comprehensive molecular genetic analyses of oxytocin receptor gene (OXTR), a positional and functional candidate gene for autism.

Familial combined hyperlipidemia is associated with upstream transcription factor 1 (USF1).

Familial combined hyperlipidemia (FCHL), characterized by elevated levels of serum total cholesterol, triglycerides or both, is observed in about 20% of individuals with premature coronary heart disease. We previously identified a locus linked to FCHL on 1q21-q23 in Finnish families with the disease. This region has also been linked to FCHL in families from other populations as well as to type 2 diabetes mellitus. These clinical entities have several overlapping phenotypic features, raising the possibility that the same gene may underlie the obtained linkage results. Here, we show that the human gene encoding thioredoxin interacting protein (TXNIP) on 1q, which underlies combined hyperlipidemia in mice, is not associated with FCHL. We show that FCHL is linked and associated with the gene encoding upstream transcription factor 1 (USF1) in 60 extended families with FCHL, including 721 genotyped individuals (P = 0.00002), especially in males with high triglycerides (P = 0.0000009). Expression profiles in fat biopsy samples from individuals with FCHL seemed to differ depending on their carrier status for the associated USF1 haplotype. USF1 encodes a transcription factor known to regulate several genes of glucose and lipid metabolism.

Effect of arsenite, maternal age, and embryonic sex on spina bifida, exencephaly, and resorption rates in the splotch mouse.

BACKGROUND: This study examines interactions of a mutation in Pax3, embryonic sex, advanced maternal age, and arsenite exposure in the splotch (Sp) mouse model, with the aim of describing gene-environment interactions for neural tube defects and embryonic lethality. METHODS: Splotch heterozygous C57BL/6J mice were crossed to produce offspring of three genotypes with a common maternal genotype that were exposed to either sodium arsenite on gestational day (GD) 8.0, or advanced maternal age (dams older than 12 months). Embryos were extracted on GD 12 and genotyped for both Pax3 and sex. RESULTS: Arsenite treatment was a significant contributor to both exencephaly and spina bifida. Advanced maternal age resulted in a high exencephaly rate in Sp/Sp female embryos (but not other genotypes) and a high overall resorption rate. Arsenite treatment and advanced maternal age resulted in elevated sex ratios (male:female) for heterozygous and wild-type embryos. The sex ratio was highest for wild-type embryos and was lowered as the number of mutant Pax3 alleles increased. The sex ratio was not significantly different from 1.0 for splotch homozygotes. Control litters had spina bifida rates that were 95% in homozygous, 6% in heterozygous, and 0% in wild-type embryos. CONCLUSIONS: If arsenite produces exencephaly by inactivating the Pax3 protein, then the fact that the exencephaly rate was increased in Sp/Sp embryos with no functional Pax3 indicates that arsenite may either induce this defect through additional pathways, or may alter the response via modifier genes. Genetic and environmental factors contributed to the determination of murine sex ratios, with female embryos being more susceptible to loss.

Familiality and co-occurrence of clinical features of systemic lupus erythematosus.

OBJECTIVE: To evaluate familiality of 15 clinical and laboratory features in systemic lupus erythematosus (SLE)-affected sibpairs, and to estimate correlations with the age at SLE diagnosis in affected sibpairs and parent-offspring pairs. METHODS: Concordance rates and sibling risk ratios were used as indicators of familiality for 15 manifestations of SLE. Pearson's correlations and paired t-tests were used to compare the age at SLE diagnosis in affected sibpairs and in parent-offspring pairs. RESULTS: Increased sibling risk ratios (1.9-3.9) for thrombocytopenia, discoid rash, neurologic disorder (defined as seizure or psychosis), and hemolytic anemia were observed in 159 SLE-affected sibpairs. Among these clinical features, paired expression of hemolytic anemia plus thrombocytopenia and hemolytic anemia plus neurologic disorder appeared to be more frequent in 709 SLE patients than would be expected by chance (P < 0.00001 and P < 0.007, respectively). The ratio of the presence of both hemolytic anemia and neurologic disorder was approximately 13 times higher in the younger affected sib than in the older affected sib (P < 0.02). Familiality of patient age at SLE diagnosis, as observed by relative correlations, was greater in 125 affected sibpairs (r = 0.67, P < 0.0001) than in 37 affected parent-offspring pairs (r = 0.47, P = 0.003). The median +/- SD age at SLE diagnosis was significantly lower in offspring (21.5 +/- 10.1 years) than in their parents (41.6 +/- 15.8 years) (P < 0.0001) but was not different in sibpairs. The combined non-Caucasian sibpairs had a younger mean age at SLE diagnosis compared with Caucasian sibpairs (P = 0.014). CONCLUSION: Evidence for familiality of thrombocytopenia, discoid rash, neurologic disorder, hemolytic anemia, and co-occurring neurologic disorder plus hemolytic anemia in SLE was observed in 159 affected sibpairs. Familiality of the age at SLE diagnosis in relative pairs suggests that shared genes and/or shared environmental exposures impact disease susceptibility. Shared immediate environmental triggers appear less compelling, because the average time between dates of diagnosis was 11 years in parent-offspring pairs and 7.5 years in affected sibpairs. The significantly earlier age at disease diagnosis in offspring compared with their parents suggests that some forms of anticipation might play a role in susceptibility to SLE. Stratifying families by subphenotypes that are familial may reduce heterogeneity and facilitate identification of genetic risk factors for SLE.

Evidence that Smith-McCort dysplasia and Dyggve-Melchior-Clausen dysplasia are allelic disorders that result from mutations in a gene on chromosome 18q12.

Smith-McCort dysplasia is a rare autosomal recessive osteochondrodysplasia characterized by short limbs and a short trunk with a barrel-shaped chest. The radiographic phenotype includes platyspondyly, generalized abnormalities of the epiphyses and metaphyses, and a distinctive lacy appearance of the iliac crest. We performed a genomewide scan in a consanguineous family from Guam and found evidence of linkage to loci on chromosome 18q12. Analysis of a second, smaller family was also consistent with linkage to this region, producing a maximum combined two-point LOD score of 3.04 at a recombination fraction of 0 for the marker at locus D18S450. A 10.7-cM region containing the disease gene was defined by recombination events in two affected individuals in the larger family. Furthermore, all affected children in the larger family were homozygous for a subset of marker loci within this region, defining a 1.5-cM interval likely to contain the defective gene. Analysis of three small, unrelated families with Dyggve-Melchior-Clausen syndrome, a radiographically identical disorder with the additional clinical finding of mental retardation, provided evidence of linkage to the same region, a result consistent with the hypothesis that the two disorders are allelic.

Palmar hyperhidrosis: evidence of genetic transmission.

BACKGROUND: Primary palmar hyperhidrosis is a condition marked by excessive perspiration and is reported to have an incidence of 1% in the Western population. It is a potentially disabling disorder that interferes with social, psychological, and professional activities. Over the past several years, several investigators have reported a positive family history in their patients treated for hyperhidrosis. To date, the cause is unknown; furthermore, epidemiologic data are scarce and inadequate. METHODS: To characterize the genetic contribution to hyperhidrosis, we conducted a prospective study of 58 consecutive patients with palmar, plantar, or axillary hyperhidrosis treated with thoracoscopic sympathectomy from September 1993 to July 1999. Forty-nine of the 58 probands volunteered family history data for these analyses (84% response rate). A standardized questionnaire was administered during the postoperative visit or by phone interview, and a detailed family history was obtained. The same questionnaire was also administered to a set of 20 control patients. The familial aggregation of hyperhidrosis has been quantified by estimating the recurrence risks to the offspring, parents, siblings, aunts, uncles, and cousins of 49 probands and 20 controls. We estimated the penetrance by use of a genetic analysis program. RESULTS: Thirty-two of 49 (65%) reported a positive family history in our hyperhidrosis group, and 0% reported a positive family history in our control group. A recurrence risk of 0.28 in the offspring of probands compared with frequency of 0.01 in the general population provides strong evidence for vertical transmission of this disorder in pedigrees and is further supported by the 0.14 risk to the parents of the probands. The results indicate that the disease allele is present in about 5% of the population and that one or two copies of the allele will result in hyperhidrosis 25% of the time, whereas the normal allele will result in hyperhidrosis less than 1% of the time. CONCLUSIONS: We conclude that primary palmar hyperhidrosis is a hereditary disorder, with variable penetrance and no proof of sex-linked transmission. However, this does not exclude other possible causes, and we anticipate that genetic confirmation of this disorder may lead to earlier diagnoses and advances in medical and psychosocial interventions.